Bridging inhaled aerosol dosimetry to physiologically based pharmacokinetic modeling for toxicological assessment: nicotine delivery systems and beyond.

One of the challenges for toxicological assessment of inhaled aerosols is to accurately predict their deposited and absorbed dose. Transport, evolution, and deposition of liquid aerosols are driven by complex processes dominated by convection-diffusion that depend on various factors related to physics and chemistry. These factors include the physicochemical properties of the pure substance of interest and associated mixtures, the physical and chemical properties of the aerosols generated, the interplay between different factors during transportation and deposition, and the subject-specific inhalation topography. Several inhalation-based physiologically based pharmacokinetic (PBPK) models have been developed, but the applicability of these models for aerosols has yet to be verified. Nicotine is among several substances that are often delivered via the pulmonary route, with varied kinetics depending upon the route of exposure. This was used as an opportunity to review and discuss the current knowledge and state-of-the-art tools combining aerosol dosimetry predictions with PBPK modeling efforts. A validated tool could then be used to perform for toxicological assessment of other inhaled therapeutic substances. The Science Panel from the Alliance of Risk Assessment have convened at the "Beyond Science and Decisions: From Problem Formulation to Dose-Response Assessment" workshop to evaluate modeling approaches and address derivation of exposure-internal dose estimations for inhaled aerosols containing nicotine or other substances. The discussion involved PBPK model evaluation criteria, challenges, and choices that arise in such a model design, development, and application as a computational tool for use in human toxicological assessments.

The impact of genome evolution on the allotetraploid Nicotiana rustica - an intriguing story of enhanced alkaloid production.

BACKGROUND: Nicotiana rustica (Aztec tobacco), like common tobacco (Nicotiana tabacum), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n = 4x = 48) with its three putative diploid progenitors (2.3-3 Gb; 2n = 2x =24), Nicotiana undulata, Nicotiana paniculata and Nicotiana knightiana. RESULTS: In total, 41% of N. rustica genome originated from the paternal donor (N. undulata), while 59% originated from the maternal donor (N. paniculata/N. knightiana). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata. Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica. Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata, and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot. CONCLUSIONS: The availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.

Systems toxicology meta-analysis of in vitro assessment studies: biological impact of a candidate modified-risk tobacco product aerosol compared with cigarette smoke on human organotypic cultures of the aerodigestive tract.

Systems biology combines comprehensive molecular analyses with quantitative modeling to understand the characteristics of a biological system as a whole. Leveraging a similar approach, systems toxicology aims to decipher complex biological responses following exposures. This work reports a systems toxicology meta-analysis in the context of in vitro assessment of a candidate modified-risk tobacco product (MRTP) using three human organotypic cultures of the aerodigestive tract (buccal, bronchial, and nasal epithelia). Complementing a series of functional measures, a causal network enrichment analysis of transcriptomic data was used to compare quantitatively the biological impact of aerosol from the Tobacco Heating System (THS) 2.2, a candidate MRTP, with 3R4F cigarette smoke (CS) at similar nicotine concentrations. Lower toxicity was observed in all cultures following exposure to THS2.2 aerosol compared with 3R4F CS. Because of their morphological differences, a smaller exposure impact was observed in the buccal (stratified epithelium) compared with the bronchial and nasal (pseudostratified epithelium). However, the causal network enrichment approach supported a similar mechanistic impact of CS across the three cultures, including the impact on xenobiotic, oxidative stress, and inflammatory responses. At comparable nicotine concentrations, THS2.2 aerosol elicited reduced and more transient effects on these processes. To demonstrate the benefits of additional data modalities, we employed a newly established targeted mass-spectrometry marker panel to further confirm the reduced cellular stress responses elicited by THS2.2 aerosol compared with 3R4F CS in the nasal culture. Overall, this work demonstrates the applicability and robustness of the systems toxicology approach for in vitro inhalation toxicity assessment.

Identification of gene expression signature for cigarette smoke exposure response--from man to mouse.

Gene expression profiling data can be used in toxicology to assess both the level and impact of toxicant exposure, aligned with a vision of 21st century toxicology. Here, we present a whole blood-derived gene signature that can distinguish current smokers from either nonsmokers or former smokers with high specificity and sensitivity. Such a signature that can be measured in a surrogate tissue (whole blood) may help in monitoring smoking exposure as well as discontinuation of exposure when the primarily impacted tissue (e.g., lung) is not readily accessible. The signature consisted of LRRN3, SASH1, PALLD, RGL1, TNFRSF17, CDKN1C, IGJ, RRM2, ID3, SERPING1, and FUCA1. Several members of this signature have been previously described in the context of smoking. The signature translated well across species and could distinguish mice that were exposed to cigarette smoke from ones exposed to air only or had been withdrawn from cigarette smoke exposure. Finally, the small signature of only 11 genes could be converted into a polymerase chain reaction-based assay that could serve as a marker to monitor compliance with a smoking abstinence protocol.

Biological impact of cigarette smoke compared to an aerosol produced from a prototypic modified risk tobacco product on normal human bronchial epithelial cells.

Cigarette smoking causes serious and fatal diseases. The best way for smokers to avoid health risks is to quit smoking. Using modified risk tobacco products (MRTPs) may be an alternative to reduce the harm caused for those who are unwilling to quit smoking, but little is known about the toxic effects of MRTPs, nor were the molecular mechanisms of toxicity investigated in detail. The toxicity of an MRTP and the potential molecular mechanisms involved were investigated in high-content screening tests and whole genome transcriptomics analyses using human bronchial epithelial cells. The prototypic (p)MRTP that was tested had less impact than reference cigarette 3R4F on the cellular oxidative stress response and cell death pathways. Higher pMRTP aerosol extract concentrations had impact on pathways associated with the detoxification of xenobiotics and the reduction of oxidative damage. A pMRTP aerosol concentration up to 18 times higher than the 3R4F caused similar perturbation effects in biological networks and led to the perturbation of networks related to cell stress, and proliferation biology. These results may further facilitate the development of a systems toxicology-based impact assessment for use in future risk assessments in line with the 21st century toxicology paradigm, as shown here for an MRTP.

Genomic impact of cigarette smoke, with application to three smoking-related diseases.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

Tandem mass spectrometry protein identification on a PC grid.

We present a method to grid-enable tandem mass spectrometry protein identification. The implemented parallelization strategy embeds the open-source x!tandem tool in a grid-enabled workflow. This allows rapid analysis of large-scale mass spectrometry experiments on existing heterogeneous hardware. We have explored different data-splitting schemes, considering both splitting spectra datasets and protein databases, and examine the impact of the different schemes on scoring and computation time. While resulting peptide e-values exhibit fluctuation, we show that these variations are small, caused by statistical rather than numerical instability, and are not specific to the grid environment. The correlation coefficient of results obtained on a standalone machine versus the grid environment is found to be better than 0.933 for spectra and 0.984 for protein identification, demonstrating the validity of our approach. Finally, we examine the effect of different splitting schemes of spectra and protein data on CPU time and overall wall clock time, revealing that judicious splitting of both data sets yields best overall performance.

MDB: a database system utilizing automatic construction of modules and STAR-derived universal language.

MOTIVATION: The value of information greatly increases if stored in databases. The objective was to construct a multi-purpose database system primarily designed to store and provide access to three-dimensional structures of biological molecules including theoretical models. RESULTS: A dictionary defining data format and structure for three-dimensional models of biological molecules (MDB dictionary) was developed. The dictionary was written using universal, standardized data description language. This language can be applied to describe data with no restrictions on their origin or type, including metadata. Thus both the data definitions (format) and database descriptions are created using the uniform language and processed with universal software. A database and data design technique that allowed use of dictionaries to automatically construct relational databases was developed. This technique was employed to construct the MDB database system. Data design developed and applied in the MDB project makes it possible to carry out data curation utilizing the database engine to identify errors. It also allows storage and query of data at different levels of consistency with the standard format specifications, i.e. both the correctly formatted data, and data that requires further curation. AVAILABILITY: The MDB dictionary is available at http://www.gwer.ch/proteinstructure/mdb and as part of the PDB resources at http://pdb.rutgers.edu/mmcif/.

Protein structure computing in the genomic era.

Functional analysis of the proteins discovered in fully sequenced genomes represents the next major challenge of life science research. Computational methods play a crucial role in this activity and, among them, comparative protein modelling is of great assistance during the rational design of mutagenesis experiments. Our aim over the last several years has been to further the use of 3-D model structures in this field. Therefore, we have developed a comparative protein modelling environment composed of the Swiss-PdbViewer (sequence to structure workbench and viewing program), SWISS-MODEL (internet-based server for model generation) and a database of a model generated with 3DCrunch.

Automated protein modelling--the proteome in 3D.

Functional analysis of the proteins discovered in fully sequenced genomes represent the next major challenge of life science research. Computational methods play an increasingly important role in this activity. Among them, comparative protein modelling will play a major role in this challenge, especially in the light of the Structural Genomics programmes about to be started around the world. In recent years, much progress has been made in automating these methods, enabling the production of models for genome scale problems. In this review we discuss how protein models can be applied to functional analysis, as well as some of the current issues and limitations inherent to these methods.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

New insulin-like proteins with atypical disulfide bond pattern characterized in Caenorhabditis elegans by comparative sequence analysis and homology modeling.

We have identified three new families of insulin homologs in Caenorhabditis elegans. In two of these families, concerted mutations suggest that an additional disulfide bond links B and A domains, and that the A-domain internal disulfide bond is substituted by a hydrophobic interaction. Homology modeling remarkably confirms these predictions and shows that despite this atypical disulfide bond pattern and the absence of C-like peptide, all these proteins may adopt the same fold as the insulin. Interestingly, whereas we identified 10 insulin-like peptides, only one insulin-like-receptor (daf-2) has been found. We propose that these insulin-related peptides may correspond to different activators or inhibitors of the daf-2 insulin-regulating pathway.

Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines.

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to the macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7%) and MIP-1alpha (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes, T-lymphocytes and, to a lesser degree, eosinophils at nanomolar concentrations; it has no effect on neutrophil migration. In receptor-binding assays, MIP-5 shows IC50 values of 12 nM for competition with 125I-MIP-1alpha for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for binding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 was only able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3.

Cloning of the TMPRSS2 gene, which encodes a novel serine protease with transmembrane, LDLRA, and SRCR domains and maps to 21q22.3.

To contribute to the development of the transcription map of human chromosome 21 (HC21), we have used exon trapping from pools of HC21-specific cosmids. Using selected trapped exons, we have identified a novel gene (named TMPRSS2) that encodes a multimeric protein with a serine protease domain. The TMPRSS2 3.8-kb mRNA is expressed strongly in small intestine and weakly in several other tissues. The full-length cDNA encodes a predicted protein of 492 amino acids that contains the following domains: (i) A serine protease domain (aa 255-492) of the S1 family that probably cleaves at Arg or Lys residues. (ii) An SRCR (scavenger receptor cysteine-rich) domain (aa 149-242) of group A (6 conserved Cys). This type of domain is involved in the binding to other cell surface or extracellular molecules. (iii) An LDLRA (LDL receptor class A) domain (aa 113-148). This type of domain forms a binding site for calcium. (iv) A predicted transmembrane domain (aa 84-106). No typical signal peptide was recognized. The gene was mapped to 21q22.3 between markers ERG and D21S56 in the same P1 as MX1. The physiological role of TMPRSS2 and its involvement in trisomy 21 phenotypes or monogenic disorders that map to HC21 are unknown.

Spatial orientation of the alpha and betac receptor chain binding sites on monomeric human interleukin-5 constructs.

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R. R., and Huston, D. P. (1996) Nature 379, 652-655). We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer. This was achieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge onto these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three other helices. The proteins dimerize with Kd values ranging from 20 to 200 microM and are therefore monomeric at the picomolar concentrations where they are biologically active. Introduction of a second disulfide confers increased stability, but this increased rigidity results in lower activity of the protein. Contrary to wild type IL-5, mutation of the betac contact residue on the first helix, Glu12, to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities. These results indicate that activation of the IL-5 receptor complex is not mediated solely by Glu12 on the first helix, and alternative mechanisms are discussed.

Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.

Clinical spectrum of X-linked hyper-IgM syndrome.

We report the clinical and immunologic features and outcome in 56 patients with X-linked hyper-IgM syndrome, a disorder caused by mutations in the CD40 ligand gene. Upper and lower respiratory tract infections (the latter frequently caused by Pneumocystis carinii), chronic diarrhea, and liver involvement (both often associated with Cryptosporidium infection) were common. Many patients had chronic neutropenia associated with oral and rectal ulcers. The marked prevalence of infections caused by intracellular pathogens suggests some degree of impairment of cell-mediated immunity. Although lymphocyte counts and in vitro proliferation to mitogens were normal, a defective in vitro proliferative response to antigens was observed in some patients, and additional defects of cell-mediated immunity may be presumed on the basis of current knowledge of CD40-ligand function. All patients received regular infusions of immunoglobulins. Four patients underwent liver transplantation because of sclerosing cholangitis, which relapsed in there. Three patients underwent bone marrow transplantation. Thirteen patients (23%) died of infection and/or liver disease. X-linked hyper-IgM syndrome, once considered a clinical variant of hypogammaglobulinemia, is a severe immunodeficiency with significant cellular involvement and a high mortality rate.